The more specific biological functions of sialic acids appear to depend upon diversity generated by different linkages to the underlying sugar chain, and by modifications such as O-acetylation. Alpha-2-6-linked sialic acids are present in B and T-cells, endothelial cells and hepatocytes. This sequence generates ligands for CD22, a B cell-specific member of the new family of "I-type lectins of the immunoglobulin superfamily. These ligands can be masked by O-acetylation of the sialic acids. Alpha-2-8-linked sialicacids on gangliosides can also be selectively O-acetylated in melanoma cells, neuroectodermal tissues, and certain leucocytes. Biosynthesis of these sequences involves specific sialyltransferases and O-acetyltransferase(s) - while some of the former have been purified and cloned, the latter have not. Ablation of 9-O- acetylation by Influenza C hemagglutinin-esterase (CHE) can help to elucidate functions of this modification. A recombinant soluble form of CHE has been developed for this purpose, and can be modified to probe for 9-O-acetylation in cells and tissues. Human melanoma gangliosidesialic acids can also be de-N-acetylated, and the tyrosine kinase inhibitor genistein increases expression of this modification. We now propose to focus upon the following: 1. Compare the tissue distribution of 9-O-acetylation, among four mammalian species.This is made possible by a CHE-based probe. O- acetylation should be conserved in tissues where it is of broad relevance. 2. Cell cycle dependence of 9-O-acetylation and de-N-acetylation of sialic acids. Preliminary studies indicate that these modifications my be regulated in a cell-cycle-dependent manner. 3. Attempts at molecular cloning of the sialic acid-specific O- acetyltransferase(s). All attempts to purify these enzymes have failed - expression cloning will be attempted using multiple approaches. 4.Study the mechanisms of de- and re-N-acetylation of sialic acids in melanoma cells. If time allows, we will pursue the enzymatic mechanisms by which de-N-acetylation occurs with genstein treatment. 5. Study the effect of B-cells activation on CD22, CD22 ligands and O- acetylation. Polyclonally activated B-cells will be studied for changes in the expression and connection of CD22 and its ligands. 6. Compare sialic acid recognition by human CD22beta with that by mouse CD22. This comparative study is critical for the planning of the aim #7. 7. Use sialyloligosaccharides or 9-de-O-acetylation to perturb T-B cell interactions in vitro, and in vivo. The outcome will be studied both in terms of tissue morphology, as well as the immune response. 8. Abrogate CD22 expression in the mo use by gene disruption and/or tissue-specific gene deletion. This will provide critical in formation regarding the role of CD22 lectin in the immune response. 9. Study the structure of CD22 and CHIC in complex with cognate oligosaccharides. Recombinant CD22 and CHE will be used to obtain a crystal structure, and to define the interaction-with sialic acids. 10.Ultrastructurally localize the previously described "cytosolic" CD22. This pool of CD22 persists almost throughout the life span of a B- lymphocyte. Its location has an important bearing on CD22 function.